Glioblastomas is the most malignant form of primary gliomas. Despite the advance in medicine, it remains refractory to conventional therapies. Recent progress in understanding of molecular events leading to glioma genesis provides alternative therapeutic targets for management of gliomas. Several novel small molecules targeting various signaling nodes along receptor tyrosine kinases (RTKs) and PTEN-PI3K-akt pathway have been developed and are currently in different stages of testing, including EGFR inhibitors like Tarceva, Iressa, and AEE788 as well as mTOR inhibitors such as Rapamycin and CCI-779. However, they have met with only limited success in early phase l/ll clinical trials when used alone, most likely because other compensatory or collateral pathways negate the therapeutic efficacy of inhibiting a single signal node. The logical next step, is to identify drugs that would block these pathways so that when given in combination, they elicit enhanced anti-tumor effect. Our long-term goal is to identify specific molecular agents that, when used in combination, would improve the therapeutic outcome for glioblastoma. This proposal seeks to use modern siRNA gene-silencing techniques to identify targets whose inactivation when combined with small molecule inhibitors would confer synergistic anti-tumor activity. Following are specific aims to achieve this goal. Specific Aim 1: To identify the molecular targets or pathways whose disruption in combination with lead drug treatment will result in a lethal phenotype. Our working hypothesis is that silencing overlapping pathways or compensatory activated in response to treatment with a small molecule inhibitor (lead drug) will result in cell death. Using both a siRNA library and a signaling pathway drug, a genome-wide microarray screen will be employed to identify targets or pathways that when silenced will sensitize cells to drug-induced cell death. We expect to identify one or more targets for each of the four drugs tested. Specific Aim 2: To validate the targets or pathways whose inactivation sensitizes tumor cells to drug-induced cell death in vitro. Our working hypothesis is that inhibiting the targets identified in aim 1 will synergistically enhance the anti-tumor effect of the lead drug in vitro. Targets identified in aim 1 will be validated by 1) combining inactivation of targets by individual siRNA with lead drug treatment;2) combining inactivation of targets with commercially available drug and lead drug treatment;3) combining inactivation of targets by a dominant-negative constructs with lead drug treatment;. 4) assessment of lead drug effect on a battery of glioma cell lines;and 5) In vitro target evaluation with reverse phase protein lysate array (RPPA). Specific Aim 3: To evaluate the combined therapeutic efficacy of lead drug and target gene inactivation in vivo. Our working hypothesis is that one or more of the targets validated in vitro will exert drug-synergy when tested in animal models. The in vivo therapeutic efficacy of drug treatment with target gene inactivation will be tested in both intracranial and subcutaneous animal models. Tumor cells stably expressing siRNA against the target gene will be implanted into animals followed by lead drug treatment. For those targets whose inactivation could be achieved by using small molecules, animals receiving unmodified tumor cells will be treated with drug and small molecules. Tumor size and animal survival will be assessed to evaluate the therapeutic efficacy of combination treatments. The activity of downstream effector genes will be examined by reverse phase protein lysate array (RPPA). We expect that the results will not only advance our knowledge of the association between comp signal pathways and response to a particular targeted therapy but also increase the range of options for treating glioblastoma with combination therapy.